Glycan modifications of natural products are increasingly important in the discovery of new pharmaceuticals. While glycodiversity gives rise to important therapeutic properties of bioactive molecules, exploring glycodiversity has challenges. Isolating glycosylated compounds from nature is often difficult due to low concentrations and the presence of multiple related molecules with similar structures and properties. Chemical synthesis is challenging task and may only produce small milligram amounts. The enzymatic synthesis of glycans has many potential advantages for producing both natural and novel compounds is significantly limited by the availability of the activated NDP-sugar substrates required by glycosyltransferases involved in the glycosylation reactions. At zuChem we have engineered proprietary enzymes involved in activated sugar synthesis including sugar kinases and nucleotidyltransferase with broadened substrate specificities producing a wide range of sugar-1- phosphates and activated sugars. The enzymes are stable and carry out reactions quickly and at high titer. Despite this, however, challenges remain with their use on a rapid and broad scale. The enzymes exhibit product and substrate inhibition and run under different physiological reaction conditions. This prevents a simple one-pot method for activated sugars to be developed - intermediates need complex purification before use in the next reaction step. As a result, throughput and the ability to produce larger volumes of activated sugars is limited. Finally, the cost of nucleotides remains expensive for large-scale production of compounds. Here, we propose to develop an integrated system for simple production of a large library of natural and novel activated NDP-sugars. The system will contain enzymes engineered to eliminate substrate/product inhibition and work under similar physiological conditions. They will be utilized in a one-pot or immobilized setup with a nucleotide recycling system. Starting from the proprietary enzymes we have developed we will use a powerful screen we have designed to rapidly identify mutants with improved enzyme properties. In Phase I we will demonstrate the feasibility of developing the system. We will show that a polyphosphate kinase can be engineered to tolerate higher levels of the ADP co-product, and a nucleotidyltransferase can be engineered with decreased sensitivity to nucleotides. In Phase II we will further improve the enzymes, optimize mutation combinations and optimize the cofactor recycling system. We will also develop one-pot and/or enzyme immobilization methods to create an integrated system for the rapid production of activated sugars. The system will be able to produce not only activated sugars, but custom glycosylated products when fed only sugar, catalytic amounts of nucleotide-triphosphates (NTP), polyphosphate, and the customer?s desired glycosyltransferase and substrate. At preparative and industrial scales the system will be adapted for the production of important oligosaccharides. In Phase III we will commercialize the system by selling or licensing it for use to researchers, and selling custom activated sugars, oligosaccharides and other glycosylated compounds.